Antibody Microarray: A Technique to Study the Protein Expression of miRNA Treated Lung Cancer Cells

1. miR-143 and miR-506 transfection

CAUTION: Use latex gloves, protective eyeglasses, and a laboratory coat while performing the described experiments. When required, use the biosafety cabinet with the cabinet fan on, without blocking the airways or disturbing the laminar airflow. Always set the protecting glass window to the appropriate height, as described by the manufacturer.

1. Seed NSCLC A549 cells in a T25 cm² flask/6/96 well plate in DMEM/F12K media supplemented with 10% FBS and 1% penicillin-streptomycin (culture media) in a tissue culture hood and incubate overnight at 37 °C with 5% CO₂ in a tissue culture incubator.
2. Suspend miR-143 and/or miR-506 mimics, or scramble siRNA with transfecting agent (2.4 µg of miR were mixed with 14 µL of transfecting agent; see Table of Materials) in 500 µL of transfection media and 1.5 mL of serum and antibiotic-free DMEM/F12K media at a final miRNA concentration of 100 nM. miRNA amount may require optimization on different cells and concentrations. Appropriate approaches include the transfection of cells with increasing concentrations of miRNA (i.e., 50-200 nM) and evaluation of expression downregulation of the genes of interest.
3. Remove culture media from flask/plate and wash once with 1x PBS.
4. Add miRNA/scramble-transfecting agent complexes and incubate at 37 °C with 5% CO₂ for 6 h (flask size defines the added incubation volume).
5. Replace the media with 4 mL of culture media and incubate cells for 24 h and/or 48 h.

2. Protein expression by antibody cell cycle microarray

1. Seed 5 x 10⁵ cells for each sample in T25 cm² flasks and perform a transfection according to the protocol described in section 1, steps 1-5.
2. After 24 h and 48 h, harvest cells by trypsinization.
3. Transfer cell suspensions to 15 mL tubes and label them accordingly.
4. Centrifuge at 751 x g for 5 min and discard the supernatant.
5. Add 2 mL of ice-cold 1x PBS, vortex and centrifuge at 751 x g for 5 min. Discard the supernatant.
6. Repeat the washing step with 1x PBS.
7. Add 150 µL of lysis buffer supplemented with protease inhibitors. Pipet up and down gently to disrupt the cell membranes.
8. To prevent any lysis buffer interference, perform a buffer exchange to replace the lysis buffer with labeling buffer, using the manufacturer's solvent exchanging columns.
9. Quantify the total protein with a BCA assay.
10. Take 70 µg of protein sample and add labeling buffer to achieve a final volume of 75 µL.
11. Add 100 µL of dimethylformamide (DMF) to 1 mg of biotin reagent (biotin/DMF).
12. Add 3 µL of the biotin/DMF to each protein sample (biotinylated protein sample) and incubate for 2 h at RT.
13. Add 35 µL of stop reagent and mix by vortexing.
14. Incubate samples at RT for 30 min.
15. Remove the microarray slides from the refrigerator so that they warm to RT for 1 h before use.
16. Perform blocking for non-specific binding by incubating the slides with 3% dry milk solution (in blocking reagent provided by manufacturer) in a Petri dish with continuous shaking for 45 min at RT.
17. Wash the slides with ddH₂O water (unless otherwise specified, washing takes place with ddH₂O).
18. Repeat the washing step ~10x to completely remove the blocking solution from the slide surfaces. This is important to achieve a uniform and low background.
19. Remove excessive water from the slide surfaces and proceed to the next step without letting the slides dry.
20. Prepare coupling solution by dissolving 3% dry milk in a coupling reagent.
21. Add 6 mL of the coupling solution and to the full quantity of the previously prepared biotinylated protein sample from step12.
22. Place one slide in one well of the coupling chamber provided by the vendor and add ~6 mL of protein coupling mix to it.
    NOTE: Ensure that the slide is completely submerged in protein coupling mix solution.
23. Cover the coupling chamber and incubate for 2 h at RT with continuous agitation in an orbital shaker.
24. Transfer the slides to a Petri dish and add 30 mL of 1x washing buffer. Place the Petri dish in orbital shaker, shake for 10 min, and discard the solution.
25. Repeat step 24 2x.
26. Rinse the slides with ddH₂O water extensively as described in steps17 and 18 and proceed to next step immediately to avoid drying.
27. Add 30 µL of Cy3-streptavidin (0.5 mg/mL) in 30 mL of detection buffer.
28. Place the slide in a Petri dish and add 30 mL of detection buffer containing Cy3-streptavidin.
29. Incubate in an orbital shaker for 20 min with continuous shaking protected from light.
    NOTE: Cy-3 is a fluorescent dye. Cover with aluminum foil or operate under dark conditions to maintain fluorescence intensity.
30. Perform steps 24-26 and allow the slide to dry using a gentle stream of air or placing the slide in a 50 mL conical tube and centrifuge at 1300 x g for 5-10 min.
31. Place the slide in slide holder and cover with aluminum foil.
32. Scan the slide in a microarray scanner with the appropriate excitation and emission wavelengths. In the case of Cy3, the excitation wavelength peak is at ~550 nm and emission peak at ~570 nm.
33. Analyze the data (see Table of Materials for software used).